The present invention belongs to the field of purification of nucleic acids, in aqueous medium.
A process is known according to the document WO-A-95/04140 for purifying, in aqueous medium, nucleic acids present in a sample, according to which said sample is brought into contact with a particulate system consisting of silica beads, in the presence of a chaotropic substance, and then the nucleic acids attached to the beads are separated from the final aqueous solution.
In accordance with the document F. Meunier et al., Polymers for Advanced Technologies, Volume 6, pp. 489-496, (1995), the preparation of a polymer called PNIPAM, by polymerization of (1) N-isopropylacrylamide, (2) N,N-methylenebisacrylamide and (3) 2-aminoethylmethacrylate chloride, in the presence of a polymerization initiator, is described. The behavior of this surface-functionalized polymer can make it particularly suited to a covalent attachment of biological molecules.
The document EP-A-0 161 881 teaches that a heat-sensitive polymer such as the polymers obtained by copolymerization of monomers of N-alkyl- or of N-alkylene-acrylamide or methacrylamide and of monomers of acrylic or methacrylic derivatives, can be used in the isolation of biological material, by virtue of its capacity to change structure as a function of the temperature. It has an open structure at low temperature, which facilitates the attachment of a biological material, and a retracted structure at high temperature, which allows the liberation of the attached biological material. The control of the steps of attachment and liberation of the biological material can therefore be performed by varying the temperature. For a better control, it is possible, in addition, to vary the pH.
The use proposed by this document extends to the isolation of any biological material present in a sample, and in particular nucleic material and protein material, without any specificity.
According to the invention, a process for the selective isolation of a nucleic material present in a sample is provided. Even if the sample is complex and contains a protein material and/or inhibitors of enzymatic reaction, the process of the invention limits or even eliminates any isolation of the protein material and/or of said inhibitors, while promoting the isolation of the nucleic material.
A process for the isolation in aqueous phase, according to the invention, of a nucleic material present in a sample, comprises the following steps:
according to a so-called step (a) for producing the adsorption reagent, an adsorption reagent is available which comprises a sol consisting of an aqueous continuous phase and a discontinuous phase of the particulate support which comprises a functionalized, particulate polymer, said polymer being obtained by polymerization of (1) a first water-soluble monomer of acrylamide or of an acrylamide derivative, (2) at least one cross-linking agent and (3) at least a second cationic and water-soluble functional monomer, said polymer having a predetermined lower critical solubility temperature (LCST) which is between 25 and 45xc2x0 C., preferably between 30 and 40xc2x0 C., according to a so-called step (b) for bringing into contact, the adsorption reagent is brought into contact with the sample containing the nucleic material,
according to a so-called adsorption step (c), for the bringing into contact according to (b), at least one and preferably at least two of the following parameters for the reaction medium are chosen:
pH at most equal to 7,
ionic strength at most equal to 10xe2x88x922 M,
temperature less than the LCST of the polymer,
according to a so-called separation step (d), after having optionally observed that the adsorption has taken place, the discontinuous phase and in particular that having adsorbed the nucleic material are separated from the continuous phase,
according to a so-called desorption step (e), the nucleic material is dissociated, by desorption, from the particulate support by increasing the ionic strength up to an ionic strength greater than 10xe2x88x922 M.
Advantageously, for the desorption step (e), at least one of the parameters selected from the pH and the temperature is in addition varied as follows:
increase in the pH up to a pH greater than 7,
increase in the temperature up to a temperature greater than the LCST of the polymer.
The invention also relates to a process for the isolation, in aqueous phase, of a nucleic material present in a sample, comprising a step of adsorption of said nucleic material, onto a particulate support, allowing a use as such of the nucleic material adsorbed onto the particulate support, in a subsequent analytical step. This process comprises the following steps:
according to a so-called step (a) for producing the adsorption reagent, an adsorption reagent is available which comprises a sol consisting of an aqueous continuous phase and a discontinuous phase of the particulate support which comprises a functionalized, particulate polymer, said polymer being obtained by polymerization of (1) a first water-soluble monomer of acrylamide or of an acrylamide derivative, (2) at least one cross-linking agent and (3) at least a second cationic and water-soluble functional monomer, and said polymer having a predetermined lower critical solubility temperature (LCST) which is between 25 and 45xc2x0 C.,
according to a so-called step (b) for bringing into contact, the adsorption reagent is brought into contact with the sample containing the nucleic material,
according to a so-called adsorption step (c), for the bringing into contact according to (b), an ionic strength at most equal to 10xe2x88x922 M is selected for the reaction medium,
according to a so-called separation step (d), after having optionally observed that the adsorption has taken place, the discontinuous phase is separated from the continuous phase, according to which process the desorption step is optional.
In accordance with a preferred embodiment of the latter process, according to the adsorption step (c), for the bringing into contact according to (b), at least one of the following parameters is in addition selected for the reaction medium:
pH at most equal to 7,
temperature less than the LCST of the polymer.
Of course, this process may comprise, after the separation step (d), a so-called desorption step according to which the nucleic material is dissociated, by desorption, from the particulate support by varying at least one of the parameters selected from the ionic strength, the pH and the temperature, as follows,
increase in the ionic strength up to an ionic strength greater than 10-2 M
increase in the pH up to a pH greater than 7,
increase in the temperature up to a temperature greater than the LCST of the polymer.
At least the ionic strength is advantageously varied.
The processes defined above according to the invention will be preferably carried out according to two variants related to step (a).
According to a first variant which will be illustrated in the examples, the particulate support consists of said particulate polymer, and in this case, the cross-linking agent(s) (2) are water-soluble.
According to a second variant, the particulate support comprises, in addition, an organic or inorganic core, completely or partially coated with said particulate polymer, said core not modifying the adsorption properties of the polymer in relation to said nucleic material. The core or core portion then fulfills the function of the cross-linking agent (2), it being possible to provide another cross-linking agent of the water-soluble cross-linking agent type. By way of example, the core may be a polystyrene core, and/or comprise a magnetic compound.
According to a specific and preferred embodiment of these processes, at least one probe and/or one primer capable of specifically hybridizing to the nucleic material before or after step (b) is added to the sample before step (b), or to the reaction medium after step (b), and in particular after step (c) or step (d).
In another specific embodiment, the nucleic material consists of a probe or a primer, and according to (b) and (c), the adsorption reagent is brought into contact with said nucleic material in order to obtain a hybridization reagent, and then according to (bxe2x80x2), after having optionally observed that the adsorption has taken place, and separated the hybridization reagent from the reaction medium, said hybridization reagent is brought into contact with a medium containing at least one nucleic acid or nucleic acid fragment, under suitable conditions for the hybridization or the extension of the primer.
The particulate polymer is advantageously obtained by free radical polymerization in the presence of a cationic or neutral, and water-soluble, polymerization initiator.
The first monomer (1) is preferably selected from the N-alkylacrylamides and the N,N-dialkylacrylamides, and more particularly from N-isopropylacrylamide, N-ethylmethacrylamide, N-n-propylacrylamide, N-n-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N,N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-N-n-propylacrylamide, the first monomer being preferably N-isopropylacrylamide (NIPAM).
The second functional monomer(s) (3) are preferably selected from the acrylic and methacrylic derivatives, 2-aminoethylmethacrylate chloride (AEM), the N-vinylpyridine derivatives, the trialkylammonium derivatives and the isothiouronium chloride derivatives.
Advantageously, the water-soluble cross-linking agent (2) is selected from N,N-methylenebisacrylamide (MBA), ethylene glycol dimethacrylate, and the polymerization initiator is 2,2xe2x80x2-azobisamidinopropane chloride (V50).
The separation step (d) is preferably carried out according to a technique selected from centrifugation, filtration, precipitation, sedimentation and the application of a magnetic field.
Before the separation step (d), it can be optionally observed that the adsorption reaction has occurred. By way of example, HPLC or capillary electrophoresis techniques may be used.
Before disclosing the invention in greater detail, some terms used in the present description and in the claims are defined below:
isolation of a nucleic material according to the invention is understood to mean the separation, detection of this material, the enrichment of a fraction with nucleic material, according to a specific or a specific method of isolation, in a qualitative and/or quantitative manner.
A nucleic material according to the invention is a nucleic acid, a nucleic acid fragment, a mixture of nucleic acids and/or of nucleic acid fragments, or a fraction of nucleic acids and/or of fragments of nucleic acids. Nucleic acid is understood to mean any nucleic acid, in a free form or optionally combined with proteins, regardless of its cellular, bacterial or viral origin or the like. It is either a deoxyribonucleic acid or ribonucleic acid, consisting of a stretch of natural nucleotides whose constituent elements are a sugar, a phosphate group and a nitrogen base selected from adenine, guanine, uracil, cytosine, thymine and/or of nucleotides modified in at least one of the three constituent elements; by way of example, the modification may take place at the level of the bases, generating modified bases, such as inosine, 5-methyldeoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine, and such as bases modified by a tracer detectable directly or indirectly by techniques known to persons skilled in the art, by way of example bases modified by biotin; at the level of the sugar, namely the replacement of at least one deoxyribose by a polyamide; and/or at the level of the phosphate group, for example its replacement by esters selected in particular from the diphosphate, alkyl- and arylphosphonate and alkyl- and arylphosphorothioate esters. The nucleic acid according to the invention is completely or partially single-stranded and/or double-stranded, in particular it may consist of a probe-nucleic acid, probe-nucleic acid fragment, primer-nucleic acid or primer-nucleic acid fragment duplex; the duplex may be a homoduplex or a heteroduplex.
The invention is of course applied to the isolation of fragments of nucleic acids as defined above, or oligonucleotides (ODN), of variable sizes.
The nucleic material may be of natural origin, and/or obtained by genetic recombination and/or by chemical synthesis; by way of example, it may consist of a probe or a primer.
The present invention is applied to the aspecific isolation of a fraction of nucleic acids and/or of fragments of nucleic acids, which is contained in a sample, but also to the specific isolation of a nucleic acid or a nucleic acid fragment, or of a mixture of nucleic acids or of fragments of nucleic acids, which are present in a sample.
A sample as understood according to the invention comprises any sample capable of containing a nucleic material, in particular a biological sample such as that obtained from a biological fluid, a sample of food origin. The sample consists wholly or partly of a sample, in particular it may consist of an aliquot, a dilution. The sample may or may not have been subjected to a preliminary treatment, in particular of purification or lysis in order to facilitate the liberation of the nucleic acids.
The LCST of a polymer such as that which is the subject of the present invention is in particular defined and measured by techniques described in the following documents: Hiroshi Inomata et al., Macromolecules 1994, 27, 6459-6464.
A probe is a nucleotide fragment possessing a hybridization specificity under determined conditions for forming a hybridization complex with a nucleotide fragment. A probe used within the framework of the present invention will be preferably a capture probe, without nevertheless excluding the other types of probes from this context.
Primerr according to the invention is understood to mean a probe possessing a hybridization specificity under determined conditions for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), the so-called NASBA technique (xe2x80x9cNucleic Acid Sequence-Based Amplificationxe2x80x9d) or alternatively the so-called TMA technique (Transcription Mediated Amplification), in an extension process, such as sequencing, in a reverse transcription method or the like.
Acrylamide derivative according to the invention is understood to mean a polymerizable monomer corresponding to the formula R0xe2x80x94CHxe2x95x90C(R1)xe2x80x94CONR2R3, in which R0, R1, R2 and R3 represent a group selected independently from hydrogen, aliphatic or cyclic, linear or branched lower hydrocarbon groups, nitrogen-containing heterocyclic groups such as imidazole.
The adsorption of nucleic material as understood according to the present invention is defined as follows: a nucleic material is adsorbed onto a particulate support if, after a period of contact between said material and said support, at least one of the groups belonging to the constituent components of the nucleic material is attached to the surface of the support; the adsorption results from ionic interactions and/or hydrogen bonds, and possibly hydrophobic interactions, excluding any covalent bond, between the material and the support.
Finally, functionalized polymer is understood to mean a polymer having at least one interface carrying functional groups capable of generating with groups of the constituent components of the nucleic material any one of the interactions and/or bonds involved in the adsorption phenomenon. Preferably, these functional groups are selected from NH3+; NH4+; NR3+ or R represents an aliphatic or cyclic, saturated or unsaturated hydrocarbon group, it being possible for NR3+ to represent the pyridinium group; and the isothiouronium group.